Using pyr gene lacZ fusions we have isolated, additional depressed pyrmidine mutants will be isolated by selecting fast-growing strains in minimal medium containing repressive concentrations of exogenous pyrimidine. These will be screened by a transductional test to assure that the fusion is intact and by comparing lacZ expression with uridine as a pyrimidine source that they are not uptake mutants. Mutations causing derepression of pyr gene expression will be mapped and biochemical characterization of the mutants will be initiated. Forms of carbamylphosphate synthase produced by a cold-sensitive mutant of S. typhimurium under permissive conditions (37 degrees C growth in minimal medium and 29 degrees C growth in the presence of exogenous arginine) and under restrictive conditions (20 degrees C growth in minimal medium) will be purified to homogeneity. Both forms of the homogenous enzyme will be studied with respect to: kinetic properties, C-terminal amino acid residues, and the ability to interconvert the two forms by mild conditions of denaturation. The position of the structural gene encoding nucleoside diphosphokinase will be located precisely on the map of the S. typhimurium chromosome. The physiological function of the enzyme and the relative role played by it and pyruvate kinase will be studied.